Antimicrobial polypeptide from Aspergillus niger

ABSTRACT

The present invention relates to an anti-microbial polypeptide and a DNA construct encoding said anti-microbial polypeptide and the use of said anti-microbial polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK02/00289 filed May 3, 2002, which claims priority or the benefitunder 35 U.S.C. 119 of Danish application no. PA 2001 00706 filed May 4,2001 and U.S. provisional application No. 60/289,102 filed May 7, 2001,the contents of which are fully incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a DNA construct comprising a DNAsequence derived from Aspergillus niger encoding an anti-microbialpolypeptide, and variants thereof. The invention also relates to the useof said anti-microbial polypeptide for controlling or combatingmicrobial organisms; to a composition comprising the anti-microbialpolypeptide of the invention as an active ingredient; to a method ofexpressing the anti-microbial polypeptide in a host cell; to the use ofthe anti-microbial polypeptide for the preparation of a composition forthe treatment or prophylaxis of microbial organisms for use in, e.g.,wound healing composition/products or products such as bandages, medicaldevices such as, e.g., catheters and further in anti-dandruff hairproducts.

BACKGROUND OF THE INVENTION

Recently an anti-fungal peptide from Aspergillus niger has been isolatedby Lee et al. (1999), Biochem. Biophys. Res. Commun. 263, 646-651). TheA. niger antifungal peptide is reported to inhibit the growth of yeastsincluding the pathogen C. albicans. The Lee et al. reference describesthe isolation of the peptide and the determination of the amino acidsequence of the mature peptide. The gene encoding the A. nigerantifungal peptide was not cloned, and the sequence of the primarytranslation product remains unknown. It is not known whether the primaryproduct contains signal and pro-peptide sequences, which could beessential to efficient secretion and processing of the product.

Another anti-fungal peptides from Aspergillus giganteus (Nakaya, K etal., (1990), Eur. J. Biochem. 19, p. 31-38) has been isolated andcharacterized. The A. giganteus peptide is small (51 amino acids),alkaline (pl 9.3) and contains four S—S interchain bridges(Campos-Olivas, R. et al. (1995), Biochemistry 34, 3009-3021). Otherpeptides have been isolated from the Aspergilli A. clavatus and A.giganteus A3274 (WO 94/01459).

SUMMARY OF THE INVENTION

The present invention relates to a DNA construct comprising a DNAsequence encoding a polypeptide with anti-microbial activity and the useof said polypeptide.

In the first aspect the invention relates to a DNA construct comprisinga DNA sequence encoding a polypeptide exhibiting anti-microbialactivity, which sequence comprises the nucleotide sequence shown in SEQID NO: 1 or a DNA sequence being at least 60% identical to the part ofSEQ ID NO: 1 encoding the mature anti-microbial peptide, i.e., thecoding part of SEQ ID NO: 1 (which is nucleotides 208 to 510), or afragment thereof.

In the second aspect the present invention relates to a polypeptideexhibiting anti-microbial activity encoded by

-   i) a DNA sequence of the invention,-   or which polypeptide sequence    -   a) comprises the amino acid sequence shown in SEQ ID NO: 2        (positions 1 to 58), or    -   b) a fragment and/or variant of the amino acid sequence shown in        SEQ ID NO: 2 (positions 1 to 58) exhibiting anti-microbial        activity, or    -   c) a fragment and/or variant as defined in b) which further has        an N-terminal extension in comparison to the mature part of SEQ        ID NO: 2, i.e., positions 1 to 58.

The term “variant” as used in connection with the anti-microbialpolypeptide is intended to indicate a polypeptide which is derived fromthe polypeptide having the amino acid sequence shown in SEQ ID NO: 2, ora naturally occurring variant. Typically, the variant differs from thenative anti-microbial polypeptide by one or more amino acid residues,which may have been added or deleted from either or both of theN-terminal or C-terminal end of the polypeptide, inserted or deleted atone or more sites within the amino acid sequence of the polypeptide, orsubstituted with one or more amino acid residues within, or at either orboth ends of the amino acid sequence of the polypeptide.

Preferably, the anti-microbial polypeptide of the invention comprisesthe amino acid sequences shown in SEQ ID NO: 2; or allelic variantsthereof; or a fragment thereof that has anti-microbial activity.

The term “fragment” of the polypeptide of the invention means apolypeptide, which lacks one or more amino acids in the amino and/orcarboxyl terminus of the parent amino acid sequence shown in SEQ ID NO:2, but retains anti-microbial activity. In other words, a polypeptidefragment of the invention is shorter than the parent polypeptide, in thepresent case the sequence shown in SEQ ID NO: 2 (positions 1 to 58). Thepolypeptide fragment of the invention may be protein engineered, e.g.,from SEQ ID NO. 2, using any methods known in the art or may also be anatural polypeptide with anti-microbial activity derived from amicro-organism being shorter (i.e., consisting of fewer amino acids)than the polypeptide shown in SEQ ID NO: 2. The anti-microbialpolypeptide of the invention may also be prepared synthetically. Thepolypeptide fragment of the invention may be from 1 to 10 amino acidsshorter in the N- and/or C-terminal in comparison to the above mentionedparent polypeptide shown in SEQ ID NO: 2, preferably from 1 to 7 aminoacids residues, especially from 1-5 amino acid residues.

An allelic variant denotes any of two or more alternative forms of agene occupying the same chromosomal locus, i.e., sequence homologybetween the two variants extends beyond the gene and its controlsequences.

Allelic variation arises naturally through mutation, and may result inpolymorphism within populations.

Gene mutations can be silent (no change in the encoded polypeptide) ormay encode polypeptides having altered amino acid sequences. An allelicvariant of a polypeptide is a polypeptide encoded by an allelic variantof a gene.

The amino acid sequences of homologous anti-microbial polypeptides maydiffer from the amino acid sequence shown in SEQ ID NO: 2 by aninsertion or deletion of one or more amino acid residues and/or thesubstitution of one or more amino acid residues by different amino acidresidues. Preferably, amino acid changes are of a minor nature, that isconservative amino acid substitutions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 1-15 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal Methionine residue; or smallextensions of up to about 20-25 residues that, e.g., facilitatespurification by changing net charge or another function, such as apoly-histidine tract, an antigenic epitope or a binding domain.

In the present context, the three-letter code of the amino acids hasbeen used in its conventional meaning. Unless indicated explicitly, theamino acids mentioned herein are L-amino acids.

The A. niger ORF encodes a primary translated product which can bedivided into the following functional parts: A 18 amino acids signalpeptide, a 16 amino acids pro-peptide and a 58 amino acid maturepolypeptide with anti-microbial activity.

The anti-microbial peptides of the invention are synthesized asprepro-peptides, i.e., in addition to a signal peptide the gene encodesa pro-peptide, which is matured (processed) into the active matureanti-microbial peptides.

In addition to complications seen in connected with production of mostother polypeptides, production of anti-microbial peptide have a furthercomplication in that the produced anti-microbial product may inhibit oreven kill its host cell used for the production. This is often seen as aproblem in connection with especially heterologous production.

In a third aspect, the present invention relates to a recombinantexpression vector comprising a DNA construct of the invention.

In a fourth aspect the invention related to a host cell comprising theexpression vector of the invention, in particular a cell of thefilamentous fungus genus Aspergillus, in particular of the groupAspergillus Nigri, especially the species A. niger or the groupAspergillus Flavus, especially the species A. oryzae, or the bacterialgenus Bacillus.

The present invention also relates to a method of producing ananti-microbial polypeptide of the invention, in particular comprisingthe amino acid sequence shown in SEQ ID NO: 2, or a fragment or avariant thereof exhibiting anti-microbial activity, which methodcomprises

-   (a) inserting a DNA construct encoding the polypeptide into a    suitable expression vector,-   (b) transforming a suitable host cell with the recombinant    expression vector of step (a),-   (c) culturing the transformed host cell in a suitable culture medium    under conditions conducive to the production of the polypeptide, and-   (d) recovering the polypeptide from the host cell or culture medium    obtained in step (c).

The invention also relates to a method of producing an anti-microbialpolypeptide of the invention, comprising cultivating a micro-organismwhich, in nature, is capable of producing the polypeptide on a suitableculture medium and under conditions allowing the production of thepolypeptide, and recovering the polypeptide from the resulting biomassand/or fermented culture medium.

The invention also relates to a method for homologously or recombinantlyproducing a polypeptide having anti-microbial activity of the inventioncomprising (a) cultivating the host cell of the invention underconditions conducive to expression of the polypeptide; and (b)recovering the polypeptide.

The invention also relates to a composition comprising, as an activeingredient, an anti-microbial polypeptide of the invention, whichfurther may comprise an additional biocidal agent.

The invention also relates to the use of an anti-microbial polypeptideof the invention for controlling or combating micro-organisms, such asfungal organisms or bacteria.

The composition and anti-microbial agent the invention may be used as ananti-microbial veterinarian or human therapeutic or prophylactic agent.Thus, the composition and anti-microbial polypeptide of the inventionmay be used in the preparation of a veterinarian or human therapeuticagent for the treatment of a microbial, such as fungal infection, or forprophylactic treatment.

Finally the invention relates to wound healing composition or productssuch as bandages, medical devices such as, e.g., catheters and furtherto anti-dandruff hair products, such as a shampoo.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of the amino acid sequence (SEQ ID NO: 11) ofan A. niger anti-fungal polypeptide determined by Lee et al., (1999),Biochem. Biophys. Res. Commun. 263, 646-652, the deduced amino acidsequences (SEQ ID NO: 10) for the mature anti-fungal polypeptide from A.niger (strain C-40-1), and the consensus sequence (SEQ ID NO: 12)deduced there from.

FIG. 2 shows the Standard curve for the LC-MS measurements ofAspergillus niger AMP.

FIG. 3 shows the concentration of Aspergillis niger AMP duringcultivation

DETAILED DESCRIPTION OF THE INVENTION

DNA Sequence Encoding an Anti-Microbial Polypeptide

In the first aspect the present invention relates to a DNA construct(nucleic acid construct) comprising a DNA sequence encoding apolypeptide exhibiting anti-microbial activity, such as anti-fungalactivity, which sequence comprises the nucleotide sequence shown in SEQID NO: 1 or a DNA sequence being at least 60% identical to the part ofSEQ ID NO: 1 encoding the mature anti-microbial peptide, i.e., thecoding part of SEQ ID NO: 1 (which is nucleotides 208 to 510), or afragment thereof.

The terms “nucleic acid construct/sequence” and “DNA construct/sequence”is used interchangeable in this application.

The term “DNA construct” (or “nucleic acid construct”) is defined hereinas a nucleic acid molecule, either single- or double-stranded, which isisolated from naturally occurring gene(s), which has been modified tocontain segments of nucleic acid, which are combined and juxtaposed in amanner, which would not otherwise exist in nature. The term nucleic acidconstruct may be synonymous with the term expression cassette when thenucleic acid construct contains all the control sequences required forexpression of anti-microbial polypeptide of the invention.

The term “coding sequence” as defined herein refer to the sequence,which is transcribed into mRNA and translated into a polypeptidecomprising the amino acid sequence which exhibits anti-microbialactivity of the invention when placed under the control of the belowmentioned control sequences.

The boundaries of the coding sequence are generally determined by atranslation start codon ATG at the 5′-terminus and a translation stopcodon at the 3′-terminus. A coding sequence can include, but is notlimited to, DNA, cDNA, and recombinant nucleic acid sequences.

An isolated nucleic acid sequence encoding an anti-microbial polypeptideof the invention may be manipulated in a variety of ways to provide forincreased expression. Manipulation of the nucleic acid sequence encodinga polypeptide with anti-microbial activity prior to its insertion into avector may be desirable or necessary depending on the expression vector.The techniques for modifying nucleic acid sequences utilizing cloningmethods are well known in the art.

The term “anti-microbial polypeptide” as used herein is intended tocomprise the linear as well as the active folded structures of thepolypeptide, and may, where appropriate, be used interchangeably withthe term “anti-microbial protein”.

The term “anti-microbial activity” means in the context of the presentinvention that the polypeptide encoded by the DNA sequence of theinvention is active in controlling or combating microbial organisms,including fungal organisms, such as yeasts and/or filamentous fungus,and/or bacterial organisms. Suitable assays for assessing whether apolypeptide has anti-microbial activity include the one described byLacadena, J. et al. (1995), Archives of Biochemisty and Biophysics 324(273-281) and the ones described in the “Materials & Methods” section asBioassay I.

The DNA construct comprises the DNA sequence shown in SEQ ID NO: 1, inparticular the polypeptide encoding part of SEQ ID NO: 1, or a DNAsequence being at least 60%, such as at least 70%, preferably at least80%, even more preferably at least 90%, even more preferably at least95%, even more preferred at least 97%, especially at least 99% identicalto SEQ ID NO: 1, in particular the polypeptide coding part, which may beoperably linked to one or more control sequences capable of directingthe expression of the coding sequences in a suitable host cell underconditions compatible with the control sequences.

The term “identity” (homology) used in the context of the presentinvention may be determined as the degree of identity between the twosequences indicating a derivation of the first sequence from the second.The identity may suitably be determined by means of computer programsknown in the art such as GAP provided in the GCG program package. Thus,Gap GCGv8 may be used with the following default parameters: GAPcreation penalty of 5.0 and GAP extension penalty of 0.3, defaultscoring matrix, for nucleic sequences and 3.0 and 0.1, respectively,from polypeptide sequences. GAP uses the method ofNeedleman/Wunsch/Sellers to make alignments.

A structural alignment between an anti-microbial sequence and anothersequence may be used to identify equivalent/corresponding positions inother polypeptides. One method of obtaining said structural alignment isto use the Pile Up programme from the GCG package using default valuesof gap penalties, i.e., a gap creation penalty of 3.0 and gap extensionpenalty of 0.1. Other structural alignment methods include thehydrophobic cluster analysis (Gaboriaud et al., (1987), FEBS LETTERS224, pp. 149-155) and reverse threading (Huber, T; Torda, A E, PROTEINSCIENCE Vol. 7, No. 1 pp. 142-149 (1998).

The Polypeptide Exhibiting Anti-Microbial Activity

In the second aspect the invention relates to a polypeptide exhibitinganti-microbial activity encoded by

-   i) a DNA sequence of the invention,-   or which polypeptide sequence    -   a) comprises the amino acid sequence shown in SEQ ID NO: 2        (positions 1 to 58), or    -   b) a fragment and/or variant of the amino acid sequence shown in        SEQ ID NO: 2 (positions 1 to 58) exhibiting anti-microbial        activity, or    -   c) a fragment and/or variant as defined in b) which further has        an N-terminal extension in comparison to the mature part of SEQ        ID NO: 2 (positions 1 to 58).        N-Terminal Extension

An N-terminal extension may suitably consist of from 1 to 50 aminoacids, preferably 2-20 amino acids, especially 3-15 amino acids. In oneembodiment N-terminal peptide extension does not contain an Arg (R). Inanother embodiment the N-terminal extension comprises a kex2 orkex2-like cleavage site as will be defined further below. In a preferredembodiment the N-terminal extension is a peptide, comprising at leasttwo Glu (E) and/or Asp (D) amino acid residues, such as an N-terminalextension comprising one of the following sequences: EAE, EE, DE, DD.

Kex2 Sites

Kex2 sites (see, e.g., Methods in Enzymology Vol 185, ed. D. Goeddel,Academic Press Inc. (1990), San Diego, Calif., “Gene ExpressionTechnology”) and kex2-like sites are di-basic recognition sites (i.e.,cleavage sites) found between the pro-peptide encoding region and themature region of some proteins.

Insertion of a kex2 site or a kex2-like site have in certain cases beenshown to improve correct endopeptidase processing at the pro-peptidecleavage site resulting in increased protein secretion levels.

In the context of the invention insertion of a kex2 or kex2-like siteresult in the possibility to obtain cleavage at a certain position inthe N-terminal extension resulting in an anti-microbial polypeptidebeing extended in comparison to the mature polypeptide shown in SEQ IDNO: 2 (positions 1 to 58).

In one preferred embodiment the invention relates to an anti-microbialpolypeptide produced by the method of the invention.

Expression Vector and Host Cell of the Invention

The invention also relates to a recombinant expression vector comprisinga DNA construct of the invention and a host cell comprising a DNAconstruct of the invention.

Host Cells

The host cell of the invention, either comprising a DNA construct or anexpression vector of the invention is advantageously used as a host cellin the recombinant production of an anti-microbial polypeptide of theinvention. The cell may be transformed with the DNA construct of theinvention encoding the polypeptide in question, conveniently byintegrating the DNA construct (in one or more copies) in the hostchromosome. This integration is generally considered to be an advantageas the DNA sequence is more likely to be stably maintained in the cell.Integration of the DNA constructs into the host chromosome may beperformed according to conventional methods, e.g., by homologous orheterologous recombination. Alternatively, the cell may be transformedwith an expression vector as described above in connection with thedifferent types of host cells.

The cell of the invention may be a cell of a higher organism such as amammal, an insect or a plant, but is preferably a microbial cell, e.g.,a bacterial or a fungal (including yeast) cell.

Eukaryote Host Cells

The host cell may be a eukaryote, such as a mammalian cell, an insectcell, a plant cell or a fungal cell. Useful mammalian cells includeChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, COS cells, or any number of other immortalized cell linesavailable, e.g., from the American Type Culture Collection.

In a preferred embodiment, the host cell is a fungal cell. “Fungi” asused herein includes the phyla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, In, Ainsworth and Bisby'sDictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra). Representative groups of Ascomycotainclude, e.g., Neurospora, Eupenicillium (=Penicillium), Emericella(=Aspergillus), Eurotium (=Aspergillus), and the true yeasts listedbelow. Examples of Basidiomycota include mushrooms, rusts, and smuts.Representative groups of Chytridiomycota include, e.g., Allomyces,Blastocladiella, Coelomomyces, and aquatic fungi. Representative groupsof Oomycota include, e.g., Saprolegniomycetous aquatic fungi (watermolds) such as Achlya. Examples of mitosporic fungi include Aspergillus,Penicillium, Candida, and Alternaria. Representative groups ofZygomycota include, e.g., Rhizopus and Mucor.

In a preferred embodiment, the fungal host cell is a yeast cell. “Yeast”as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). The ascosporogenous yeasts are divided into thefamilies Spermophthoraceae and Saccharomycetaceae. The latter iscomprised of four subfamilies, Schizosaccharomycoideae (e.g., genusSchizosaccharomyces), Nadsonioideae, Lipomycoideae, andSaccharomycoideae (e.g., genera Pichia, Kluyveromyces andSaccharomyces). The basidiosporogenous yeasts include the generaLeucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, andFilobasidiella. Yeasts belonging to the Fungi Imperfecti are dividedinto two families, Sporobolomycetaceae (e.g., genera Sorobolomyces andBullera) and Cryptococcaceae (e.g., genus Candida). Since theclassification of yeast may change in the future, for the purposes ofthis invention, yeast shall be defined as described in Biology andActivities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R.R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980. The biologyof yeast and manipulation of yeast genetics are well known in the art(see, e.g., Biochemistry and Genetics of Yeast, Bacil, M., Horecker, B.J., and Stopani, A. O. M., editors, 2nd edition, 1987; The Yeasts, Rose,A. H., and Harrison, J. S., editors, 2nd edition, 1987; and TheMolecular Biology of the Yeast Saccharomyces, Strathern et al., editors,1981).

In a more preferred embodiment, the yeast host cell is a cell of aspecies of Candida, Kluyveromyces, Saccharomyces, Schizosaccharomyces,Pichia, or Yarrowia. In a most preferred embodiment, the yeast host cellis a Saccharomyces carlsbergensis, Saccharomyces cerevisiae,Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyceskluyveri, Saccharomyces norbensis or Saccharomyces oviformis cell. Inanother most preferred embodiment, the yeast host cell is aKluyveromyces lactis cell. In another most preferred embodiment, theyeast host cell is a Yarrowia lipolytica cell.

In a preferred embodiment, the fungal host cell is a filamentous fungalcell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK. The filamentous fungiare characterized by a vegetative mycelium composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative. In a more preferred embodiment,the filamentous fungal host cell is a cell of a species of, but notlimited to, Acremonium, Aspergillus, Fusarium, Humicola, Mucor,Myceliophthora, Neurospora, Penicillium, Thielavia, Tolypocladium, andTrichoderma or a teleomorph or synonym thereof. In an even morepreferred embodiment, the filamentous fungal host cell is an Aspergilluscell, preferably one within the groups of Aspergillus Nigri, includingA. niger, and Aspergillus Flavus, including Asperigllus oryzae, asdefined by Raper and Fennell, (1965), pp. 71. In another even morepreferred embodiment, the filamentous fungal host cell is an Acremoniumcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Fusarium cell. In another even more preferred embodiment,the filamentous fungal host cell is a Humicola cell. In another evenmore preferred embodiment, the filamentous fungal host cell is a Mucorcell. In another even more preferred embodiment, the filamentous fungalhost cell is a Myceliophthora cell. In another even more preferredembodiment, the filamentous fungal host cell is a Neurospora cell. Inanother even more preferred embodiment, the filamentous fungal host cellis a Penicillium cell. In another even more preferred embodiment, thefilamentous fungal host cell is a Thielavia cell. In another even morepreferred embodiment, the filamentous fungal host cell is aTolypocladium cell. In another even more preferred embodiment, thefilamentous fungal host cell is a Trichoderma cell. In a most preferredembodiment the filamentous fungal host cell is within the Aspergillusgroup A. Nigri (including Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, Aspergillus niger) or the A. Flavus groupincluding Aspergillus oryzae. In another most preferred embodiment, thefilamentous fungal host cell is a Fusarium cell of the section Discolor(also known as the section Fusarium). For example, the filamentousfungal parent cell may be a Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, or Fusarium trichothecioides cell. In anotherprefered embodiment, the filamentous fungal parent cell is a Fusariumstrain of the section Elegans, e.g., Fusarium oxysporum. In another mostpreferred embodiment, the filamentous fungal host cell is a Humicolainsolens or Humicola lanuginosa cell. In another most preferredembodiment, the filamentous fungal host cell is a Mucor miehei cell. Inanother most preferred embodiment, the filamentous fungal host cell is aMyceliophthora thermophilum cell. In another most preferred embodiment,the filamentous fungal host cell is a Neurospora crassa cell. In anothermost preferred embodiment, the filamentous fungal host cell is aPenicillium purpurogenum cell. In another most preferred embodiment, thefilamentous fungal host cell is a Thielavia terrestris cell. In anothermost preferred embodiment, the Trichoderma cell is a Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei or Trichoderma viride cell.

In a preferred embodiment of the invention the host cell is a proteasedeficient or protease minus strain.

This may for be the protease deficient strain Aspergillus oryzae JaL 125having the alkaline protease gene named “alp” deleted. This strain isdescribed in WO 97/35956 (Novozymes), or EP patent no. 429,490, or theTPAP free host cell, in particular a strain of A. niger, disclosed in WO96/14404.

Transformation of Eukaryote Host Cells

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus host cells are described in EP 238 023, EP184,438, and Yelton et al., 1984, Proceedings of the National Academy ofSciences USA 81:1470-1474. A suitable method of transforming Fusariumspecies is described by Malardier et al., 1989, Gene 78:147-156 or incopending U.S. Ser. No. 08/269,449. Yeast may be transformed using theprocedures described by Becker and Guarente, In Abelson, J. N. andSimon, M. I., editors, Guide to Yeast Genetics and Molecular Biology,Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., NewYork; Ito et al., 1983, Journal of Bacteriology 153:163; and Hinnen etal., 1978, Proceedings of the National Academy of Sciences USA 75:1920.Mammalian cells may be transformed by direct uptake using the calciumphosphate precipitation method of Graham and Van der Eb (1978, Virology52:546).

Prokaryote Host Cells

The microbial host cell may be a unicellular micro-organism, e.g., aprokaryote, or a non-unicellular micro-organism, e.g., a eukaryote.Useful unicellular cells are bacterial cells such as gram positivebacteria including, but not limited to, a Bacillus cell, e.g., Bacillusalkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacilluscirculans, Bacillus coagulans, Bacillus lautus, Bacillus lentus,Bacillus licheniformis, Bacillus megaterium, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis; or aStreptomyces cell, e.g., Streptomyces lividans or Streptomyces murinus,or gram negative bacteria such as E. coli and Pseudomonas sp. In apreferred embodiment, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtiliscell.

Transformation of Prokaryote Host Cells

The transformation of a bacterial host cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168:111-115), by using competent cells (see,e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, orDubnar and Davidoff-Abelson, 1971, Journal of Molecular Biology56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6:742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169:5771-5278).

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell, which has been transformed with a gene so as to express ananti-microbial polypeptide of the invention in recoverable quantities.The anti-microbial polypeptide in question may be recovered from theplant or plant part. Alternatively, the plant or plant part containingthe recombinant anti-microbial polypeptide in question may be useddirectly for the intended use.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot).

Examples of monocot plants are grasses, such as meadow grass (bluegrass, Poa), forage grass such as festuca, lolium, temperate grass, suchas Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rapeseed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers. Also specific plant tissues, such as chloroplast, apoplast,mitochondria, vacuole, peroxisomes, and cytoplasm are considered to be aplant part. Furthermore, any plant cell, whatever the tissue origin, isconsidered to be a plant part.

Also included within the scope of the present invention are the progenyof such plants, plant parts and plant cells.

The transgenic plant or plant cell expressing an anti-microbialpolypeptide may be constructed in accordance with methods known in theart. Briefly, the plant or plant cell is constructed by incorporatingone or more expression constructs comprising a gene encoding ananti-microbial polypeptide into the plant host genome and propagatingthe resulting modified plant or plant cell into a transgenic plant orplant cell.

Conveniently, the expression construct is a nucleic acid construct,which comprises a nucleic acid sequence encoding an antimicrobialpolypeptide operably linked with appropriate regulatory sequencesrequired for expression of the nucleic acid sequence in the plant orplant part of choice. Furthermore, the expression construct may comprisea selectable marker useful for identifying host cells into which theexpression construct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences are determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the anti-microbialpolypeptide in question may be constitutive or inducible, or may bedevelopmental, stage or tissue specific, and the gene product may betargeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV promoter may be used (Francket al., 1980, Cell 21: 285-294). Organ-specific promoters may be, forexample, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889, a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941, the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93, or the aldP gene promoter from rice (Kagayaet al., 1995, Molecular and General Genetics 248: 668-674), or a woundinducible promoter such as the potato pin2 promoter (Xu et al., 1993,Plant Molecular Biology 22: 573-588.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

Transformation of Plants

The nucleic acid construct of the invention is incorporated into theplant genome according to conventional techniques known in the art,including Agrobacterium-mediated transformation, virus-mediatedtransformation, microinjection, particle bombardment, biolistictransformation, and electroporation (Gasser et al., 1990, Science 244:1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989,Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38).However it can also be used for transforming monocots, although othertransformation methods are generally preferred for these plants.Presently, the method of choice for generating transgenic monocots isparticle bombardment (microscopic gold or tungsten particles coated withthe transforming DNA) of embryonic calli or developing embryos(Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, CurrentOpinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated thereinthe expression construct are selected and regenerated into whole plantsaccording to methods well known in the art.

The present invention also relates to methods for producinganti-microbial polypeptides comprising (a) cultivating a transgenicplant or a plant cell comprising a nucleic acid sequence encoding theanti-microbial polypeptide in question under conditions conducive forproduction of the anti-microbial polypeptide in question; and (b)recovering the polypeptide in question.

Control Sequences

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for expression of thecoding sequence of the nucleic acid sequence of the invention. Eachcontrol sequence may be native or foreign to the nucleic acid sequenceencoding the polypeptide in question. Such control sequences include,but are not limited to, a leader, a polyadenylation sequence, apropeptide sequence, a promoter, a signal sequence, and a transcriptionterminator. At a minimum, the control sequences include a promoter, andtranscriptional and translational stop signals. The control sequencesmay be provided with linkers, e.g., for the purpose of introducingspecific restriction sites facilitating ligation of the controlsequences with the coding region of the nucleic acid sequence encoding apolypeptide.

Promoters

The control sequence may be an appropriate promoter sequence, a nucleicacid sequence, which is recognized by a host cell for expression of thenucleic acid sequence. The promoter sequence contains transcription andtranslation control sequences, which mediate the expression of thepolypeptide in question. The promoter may be any nucleic acid sequence,which shows transcriptional activity in the host cell of choice and maybe obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Bacterial Promoters

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, the Streptomyces coelicolor agarase gene (dagA), the Bacillussubtilis levansucrase gene (sacB), the Bacillus licheniformisalpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenicamylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene(amyQ), the Bacillus licheniformis penicillinase gene (penP), theBacillus subtilis xylA and xylB genes, and the prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of theNational Academy of Sciences USA 75:3727-3731), as well as the tacpromoter (DeBoer et al., 1983, Proceedings of the National Academy ofSciences USA 80:21-25). Further promoters are described in “Usefulproteins from recombinant bacteria” in Scientific American, 1980,242:74-94; and in J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

Fungal Promoters

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes encoding Aspergillusoryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillusniger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Rhizomucor miehei lipase, Aspergillus nidulans triose phosphateisomerase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumoxysporum trypsin-like protease (as described in U.S. Pat. No.4,288,627, which is incorporated herein by reference), and hybridsthereof. Particularly preferred promoters for use in filamentous fungalhost cells are the TAKA amylase, NA2-tpi (a hybrid of the promoters fromthe genes encoding Aspergillus niger neutral alpha-amylase andAspergillus nidulans triose phosphate isomerase), and glaA promoters.

Yeast Promoters

In a yeast host, useful promoters are obtained from the Saccharomycescerevisiae enolase (ENO-1) gene, the Saccharomyces cerevisiaegalactokinase gene (GAL1), the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP),and the Saccharomyces cerevisiae 3-phosphoglycerate kinase gene. Otheruseful promoters for yeast host cells are described by Romanos et al.,1992, Yeast 8:423-488.

Transcription Terminators

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleic acid sequence encoding the polypeptide. Anyterminator, which is functional in the host cell of choice, may be usedin the present invention.

Fungal Terminators

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes encoding Aspergillus niger neutral alpha-amylase,Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase,Aspergillus nidulans anthranilate synthase, Aspergillus nigeralpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Yeast Terminators

Preferred terminators for yeast host cells are obtained from the genesencoding Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), or Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.Terminator sequences are well known in the art for mammalian host cells.

Leader Sequences

The control sequence may also be a suitable leader sequence, anon-translated region of mRNA, which is important for translation by thehost cell. The leader sequence is operably linked to the 5′ terminus ofthe nucleic acid sequence encoding the polypeptide. Any leader sequence,which is functional in the host cell of choice, may be used in thepresent invention.

Fungus Leader Sequences

Preferred leaders for filamentous fungal host cells are obtained fromthe genes encoding Aspergillus oryzae TAKA amylase and Aspergillusoryzae triose phosphate isomerase and combinations thereof.

Yeast Leader Sequences

Suitable leaders for yeast host cells are obtained from theSaccharomyces cerevisiae enolase (ENO-1) gene, the Saccharomycescerevisiae 3-phosphoglycerate kinase gene, the Saccharomyces cerevisiaealpha-factor, and the Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase genes (ADH2/GAP).

Polyadenylation Sequences

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′ terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the host cell as a signalto add polyadenosine residues to transcribed mRNA. Any polyadenylationsequence, which is functional in the host cell of choice, may be used inthe present invention.

Fungus Polyadenylation Sequences

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes encoding Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, and Aspergillus niger alpha-glucosidase.

Yeast Polyadenylation Sequences

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15:5983-5990.Polyadenylation sequences are well known in the art for mammalian hostcells.

Signal Peptide

The control sequence may also be a signal peptide-coding region, whichcodes for an amino acid sequence linked to the amino terminus of theprotein, which can direct the expressed protein into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleic acidsequence may inherently contain a signal peptide-coding region naturallylinked in translation reading frame with the segment of the codingregion, which encodes the secreted protein. Alternatively, the 5′ end ofthe coding sequence may contain a signal peptide-coding region, which isforeign to that portion of the coding sequence, which encodes thesecreted protein. The foreign signal peptide-coding region may berequired where the coding sequence does not normally contain a signalpeptide-coding region. Alternatively, the foreign signal peptide-codingregion may simply replace the natural signal peptide-coding region inorder to obtain enhanced secretion of the protein(s) relative to thenatural signal peptide-coding region normally associated with the codingsequence. The signal peptide-coding region may be obtained from aglucoamylase or an amylase gene from an Aspergillus species, a lipase orproteinase gene from a Rhizomucor species, the gene for the alpha-factorfrom Saccharomyces cerevisiae, an amylase or a protease gene from aBacillus species, or the calf preprochymosin gene. However, any signalpeptide-coding region capable of directing the expressed protein intothe secretory pathway of a host cell of choice may be used in thepresent invention.

Bacterial Signal Peptide Sequences

An effective signal peptide-coding region for bacterial host cells isthe signal peptide-coding region obtained from the maltogenic amylasegene from Bacillus NCIB 11837, the Bacillus stearothermophilusalpha-amylase gene, the Bacillus licheniformis subtilisin gene, theBacillus licheniformis beta-lactamase gene, the Bacillusstearothermophilus neutral proteases genes (nprT, nprS, nprM), and theBacillus subtilis PrsA gene. Further signal peptides are described bySimonen and Palva, 1993, Microbiological Reviews 57:109-137.

Fungal Signal Peptide Sequences

An effective signal peptide coding region for filamentous fungal hostcells is the signal peptide coding region obtained from Aspergillusoryzae TAKA amylase gene, Aspergillus niger neutral amylase gene, theRhizomucor miehei aspartic proteinase gene, the Humicola lanuginosacellulase gene, the Candida antactica lipase B gene or the Rhizomucormiehei lipase gene.

Yeast Signal Peptide Sequences

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, Yeast 8:423-488.

Propeptide Sequences

The control sequence may also be a propeptide coding region, which codesfor an amino acid sequence positioned at the amino terminus of aprotein. The resultant protein is known as a proenzyme or propolypeptide(or a zymogen in some cases). A propolypeptide is often inactive and canbe converted to mature active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding region may be obtained from the Bacillus subtilis alkalineprotease gene (aprE), the Bacillus subtilis neutral protease gene(nprT), the Saccharomyces cerevisiae alpha-factor gene, the Candidaantactica lipase B gene, or the Myceliophthora thermophilum laccase gene(WO 95/33836).

The nucleic acid constructs of the present invention may also compriseone or more nucleic acid sequences, which encode one or more factorsthat are advantageous in the expression of the polypeptide, e.g., anactivator (e.g., a trans-acting factor), a chaperone, and a processingprotease. Any factor that is functional in the host cell of choice maybe used in the present invention. The nucleic acids encoding one or moreof these factors are not necessarily in tandem with the nucleic acidsequence encoding the polypeptide.

An activator is a protein, which activates transcription of a nucleicacid sequence encoding a polypeptide (Kudla et al., 1990, EMBO Journal9:1355-1364; Jarai and Buxton, 1994, Current Genetics 26:2238-244;Verdier, 1990, Yeast 6:271-297). The nucleic acid sequence encoding anactivator may be obtained from the genes encoding Bacillusstearothermophilus NprA (nprA), Saccharomyces cerevisiae heme activatorprotein 1 (hap1), Saccharomyces cerevisiae galactose metabolizingprotein 4 (gal4), and Aspergillus nidulans ammonia regulation protein(areA), and A. oryzae amyR. For further examples, see Verdier, 1990,supra and MacKenzie et al., 1993, Journal of General Microbiology139:2295-2307.

A chaperone is a protein, which assists another polypeptide in foldingproperly (Hartl et al., 1994, TIBS 19:20-25; Bergeron et al., 1994, TIBS19:124-128; Demolder et al., 1994, Journal of Biotechnology 32:179-189;Craig, 1993, Science 260:1902-1903; Gething and Sambrook, 1992, Nature355:33-45; Puig and Gilbert, 1994, Journal of Biological Chemistry269:7764-7771; Wang and Tsou, 1993, The FASEB Journal 7:1515-11157;Robinson et al., 1994, Bio/Technology 1:381-384). The nucleic acidsequence encoding a chaperone may be obtained from the genes encodingBacillus subtilis GroE proteins, Aspergillus oryzae protein disulphideisomerase, Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiaeBiP/GRP78, and Saccharomyces cerevisiae Hsp70. For further examples, seeGething and Sambrook et al, 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y., and Hartl et al., 1994,TIBS 19:20-25.

A processing protease is a protease that cleaves a propeptide togenerate a mature biochemically active polypeptide (Enderlin andOgrydziak, 1994, Yeast 10:67-79; Fuller et al., 1989, Proceedings of theNational Academy of Sciences USA 86:1434-1438; Julius et al., 1984, Cell37:1075-1089; Julius et al., 1983, Cell 32:839-852). The nucleic acidsequence encoding a processing protease may be obtained from the genesencoding Aspergillus niger Kex2, Saccharomyces cerevisiaedipeptidylaminopeptidase, Saccharomyces cerevisiae Kex2, and Yarrowialipolytica dibasic processing endoprotease (xpr6), tripeptidylaminopeptidase (TPAP)(WO 96/14404), and the A. oryzae dipeptidylaminopeptidase.

Regulatory Sequences

It may also be desirable to add regulatory sequences, which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems would include thelac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1system may be used. In filamentous fungi, the TAKA alpha-amylasepromoter, Aspergillus niger glucoamylase promoter, and the Aspergillusoryzae glucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those, which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene, which is amplified in the presence of methotrexate, andthe metallothionein genes, which are amplified with heavy metals. Inthese cases, the nucleic acid sequence encoding the polypeptide would beplaced in tandem with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a nucleic acid sequence of the present invention, a promoter,and transcriptional and translational stop signals. The various nucleicacid and control sequences described above may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleic acid sequence encoding the polypeptide at such sites.Alternatively, the nucleic acid sequence of the present invention may beexpressed by inserting the nucleic acid sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression, and possiblysecretion.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus), which can be conveniently subjected to recombinant DNAprocedures and can bring about the expression of the nucleic acidsequence. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. The vectors may be linear or closed circular plasmids.The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, a cosmid or an artificialchromosome. The vector may contain any means for assuringself-replication. Alternatively, the vector may be one which, whenintroduced into the host cell, is integrated into the genome andreplicated together with the chromosome(s) into which it has beenintegrated. The vector system may be a single vector or plasmid or twoor more vectors or plasmids which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

The vectors of the present invention preferably contain one or moreselectable markers, which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like. Examples of bacterial selectable markers are the dal genesfrom Bacillus subtilis or Bacillus licheniformis, or markers whichconfer antibiotic resistance such as ampicillin, kanamycin,chloramphenicol or tetracycline resistance. A frequently used mammalianmarker is the dihydrofolate reductase gene. Suitable markers for yeasthost cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Aselectable marker for use in a filamentous fungal host cell may beselected from the group including, but not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hygB (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),tipC (anthranilate synthase), and glufosinate resistance markers, aswell as equivalents from other species. Preferred for use in anAspergillus cell are the amdS and pyrG markers of Aspergillus nidulansor Aspergillus oryzae and the bar marker of Streptomyces hygroscopicus.Furthermore, selection may be accomplished by co-transformation, e.g.,as described in WO 91/17243, where the selectable marker is on aseparate vector.

The vectors of the present invention preferably contain an element(s)that permits stable integration of the vector into the host cell genomeor autonomous replication of the vector in the cell independent of thegenome of the cell.

The vectors of the present invention may be integrated into the hostcell genome when introduced into a host cell. For integration, thevector may rely on the nucleic acid sequence encoding the polypeptide orany other element of the vector for stable integration of the vectorinto the genome by homologous or none homologous recombination.Alternatively, the vector may contain additional nucleic acid sequencesfor directing integration by homologous recombination into the genome ofthe host cell. The additional nucleic acid sequences enable the vectorto be integrated into the host cell genome at a precise location(s) inthe chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 1,500 base pairs,preferably 400 to 1,500 base pairs, and most preferably 800 to 1,500base pairs, which are highly homologous with the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleic acidsequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination. These nucleicacid sequences may be any sequence that is homologous with a targetsequence in the genome of the host cell, and, furthermore, may benon-encoding or encoding sequences.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. Examples of bacterial origins of replication are theorigins of replication of plasmids pBR322, pUC19, pACYC177, pACYC184,pUB110, pE194, pTA1060, and pAMβ1. Examples of origin of replicationsfor use in a yeast host cell are the 2 micron origin of replication, thecombination of CEN6 and ARS4, and the combination of CEN3 and ARS1. Theorigin of replication may be one having a mutation which makes itsfunctioning temperature-sensitive in the host cell (see, e.g., Ehrlich,1978, Proceedings of the National Academy of Sciences USA 75:1433).

For replication in fungi the episomal replicating AMA1 plasmid vectordisclosed in WO 00/24883 may also be used.

More than one copy of a nucleic acid sequence encoding a polypeptide ofthe present invention may be inserted into the host cell to amplifyexpression of the nucleic acid sequence. Stable amplification of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome using methodswell known in the art and selecting for transformants.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al, 1989,Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor,N.Y.).

Expression of an Anti-Microbial Polypeptide of the Invention

The present invention also relates to a method of homologous orheterologous production of an anti-microbial polypeptide of theinvention in especially a bacteria or a fungal organism, especially afilamentous fungus.

More specifically the invention relates to a method of producing ananti-microbial polypeptide of the invention, in particular the aminoacid sequence shown in SEQ ID NO: 2, or a fragment or a variant thereof.The method comprises the steps of:

-   (a) inserting a DNA construct encoding the anti-microbial    polypeptide in question into a suitable expression vector,-   (b) transforming a suitable host cell with the recombinant    expression vector of step (a),-   (c) culturing the transformed host cell in a suitable culture medium    under conditions conducive to the production of the anti-microbial    polypeptide, and-   (d) recovering the polypeptide from the host cell or culture medium    obtained in step (c).

In a preferred embodiment the DNA construct is a DNA construct of theinvention, the expression vector is the vector of the invention, and/orthe host cell is a host cell of the invention (all described furtherabove).

In one embodiment the method of the invention comprises

-   (e) modifying the polypeptide, or fragment or variant obtained in    step (d).

The invention also relates to a method of homologously or recombinantlyproducing an anti-microbial polypeptide of the invention, comprisingcultivating a microorganism which, in nature, is capable of producingthe polypeptide in question on a suitable culture medium and underconditions allowing the production of the polypeptide, and recoveringthe polypeptide from the resulting biomass and/or fermented culturemedium.

The term “homologous” or “recombinant” expression or production means inthe context of the present invention that the anti-microbial polypeptidein question is expressed from a gene endogenous to the donor cell orthat a DNA construct comprising the gene encoding the anti-microbialpolypeptide (AMP) in question is introduced into the donor cell andexpressed from this genetically modified donor cell.

The term “donor cell” means the cell from which the gene encoding theanti-microbial polypeptide is obtained.

Contemplated donor microorganisms include micro-organisms such asbacteria, protozoae and algae, fungus, in particular yeasts andfilamentous fungi. Especially contemplated are filamentous fungi of thegenus Aspergillus, including Aspergillus species such as A. pallidus, A.clavatus, A. longivesica, A. rhizopodus and A. clavatonanicus, A.giganteus, and in particular of the group A. Nigria, includingAspergillus niger, Aspergillus awamori, Aspergillus foetidus,Aspergillus japonicus, A. aculeatus, A. phoenicis, A. parasiticus, andA. saitoi.

In the Example 1 cloning of a DNA sequence encoding an anti-microbialpolypeptide is obtained from a specific strain of A. niger. However, itis to be understood that a number of type strain A. niger strain may beused as the donor cell. For instance, in Example 1 the polypeptide ofthe invention may be obtained from A. niger FGSC A798.

The invention also relates to a method of heterologous production apolypeptide exhibiting anti-microbial activity of the inventioncomprising (a) cultivating the host cell of the invention underconditions conducive to expression of the polypeptide; and (b)recovering the polypeptide.

Preferred host cells are disclosed above and include especially fungalorganisms and bacteria, in particular filamentous fungi of the genusAspergillus, more particularly of the group A. Nigri, including thespecies A. niger or the group A. Flavus including A. oryzae.

The host cell may also be a bacterium of the genus Bacillus.

The term “heterologous” expression or production means that the DNAconstruct comprising the gene encoding the anti-microbial polypeptide ofthe invention is introduced into a host cell, which is different from(i.e., not the same) as the donor cell. This means that producing an A.niger anti-microbial polypeptide recombinantly in another Aspergillusniger strain is considered to be heterologous expression or production.

The inventors have succeeded in heterologous production of theanti-microbial polypeptide of the invention in filamentous fungi of thegenus Aspergillus niger and A. oryzae. When producing an anti-microbialpolypeptide of the invention in a host cell of the genus Aspergillus, inparticular of the group Aspergillus Nigri or Aspergillus Flavus highyields are obtained. For instance, in A. niger yields of secretedanti-microbial polypeptide (shown in SEQ ID NO: 2) as high as above 2.0g/L was obtained (see Example 4).

Methods of Cloning the Anti-Microbial Polypeptide

Techniques used to isolate or clone a DNA sequence comprising ananti-microbial polypeptide are known in the art and include isolationfrom genomic DNA, preparation from cDNA, or a combination thereof.

The full-length gene encoding the anti-microbial polypeptide gene mayfor instance be cloned by what is referred to as “Expression Cloning” orby cloning techniques based on conserved regions.

Obtaining an Anti-Microbial Polypeptide of the Invention

The anti-microbial polypeptide of the invention may in a preferredembodiment be obtained from a filamentous fungus, in particular a strainof the genus Aspergillus, especially A. niger.

Expression Cloning

A number of expression cloning methods are known in the art including WO99/32617. Another suitable example of such an Expression cloning methodis described in WO 93/11249 (from Novo Nordisk), which is herebyincorporated by reference. The method comprises the steps of:

-   a) cloning, in suitable vectors, a DNA library from an organism    suspected of producing one or more proteins of interest;-   b) transforming suitable yeast host cells with said vectors;-   c) culturing the host cells under suitable conditions to express any    protein of interest encoding by a clone in the DNA library; and-   d) screening for positive clones by determining any activity of a    protein expressed in step c).    Conserved Region Cloning

The cloning of the DNA sequence (nucleic acid sequence) comprising ananti-microbial polypeptide gene from genomic DNA from a strain of, e.g.,Aspergillus niger—or another organism as defined above—can be effected,e.g., by using the well-known polymerase chain reaction (PCR) orantibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleic acidsequence-based amplification (NASBA) may be used.

The term “isolated” nucleic acid sequence as used herein refers to anucleic acid sequence which is essentially free of other nucleic acidsequences, e.g., at least about 20% pure, preferably at least about 40%pure, more preferably about 60% pure, even more preferably about 80%pure, most preferably about 90% pure, and even most preferably about 95%pure, as determined by agarose gel electorphoresis. For example, anisolated nucleic acid sequence can be obtained by standard cloningprocedures used in genetic engineering to relocate the nucleic acidsequence from its natural location to a different site where it will bereproduced. The cloning procedures may involve excision and isolation ofa desired nucleic acid fragment(s) comprising the nucleic acidsequence(s) from the target filamentous fungus, insertion of thefragment into a vector, and incorporation of the recombinant vector intoa host cell where multiple copies or clones of the nucleic acid sequencewill be replicated. The nucleic acid sequence may be of genomic, cDNA,RNA, semi-synthetic, synthetic origin, or any combinations thereof.

Cloning Based on Known Genes

Known genes encoding an anti-microbial polypeptides may be used todesign an oligonucleotide probe, which can be used to isolate thefull-length genes from other organisms. Further, such probes can also beused for hybridization with the genomic or cDNA of other anti-microbialpolypeptide producing organism, following standard Southern blottingprocedures, in order to identify and isolate the corresponding orrelated anti-microbial polypeptide encoding genes.

Probes for cloning the full-length genes can be considerably shorterthan the entire sequence, but should be at least 15, preferably at least25, and more preferably at least 40 nucleotides in length. Longer probescan also be used. Both DNA and RNA probes can be used. The probes aretypically labeled for detecting the corresponding gene (for example,with ³²P, ³H, ³⁵S, biotin, or avidin). A PCR reaction using thedegenerate primers mentioned herein and genomic DNA or first-strand cDNAfrom, e.g., the filamentous fungi of the genus Aspergillus andPenicillium can also can be used to generate a probe to clone thecorresponding genomic or cDNA in other organims.

Composition of the Invention

The invention also relates to a composition comprising, as an activeingredient, an anti-microbial polypeptide of the invention as definedabove, which further may comprise an additional biocidal agent.

Use of the Anti-Microbial Polypeptide of the Invention

The invention also relates to the use of an anti-microbial polypeptideor composition of the invention as a medicament. Further, ananti-microbial polypeptide or composition of the invention may also beused for the manufacture of a medicament for controlling or combatingmicroorganisms, such as fungal organisms or bacteria.

The composition and anti-microbial polypeptide of the invention may beused as an anti-microbial veterinarian or human therapeutic orprophylactic agent. Thus, the composition and anti-microbial polypeptideof the invention may be used in the preparation of veterinarian or humantherapeutic agents or prophylactic agents for the treatment of amicrobial, such as fungal infection.

The composition of the invention comprises an effective amount of theanti-microbial polypeptide of the invention.

The term “effective amount” when used herein is intended to mean anamount of the anti-microbial polypeptide comprising the amino acidsequence shown in SEQ ID NO: 2, or a fragment or a variant thereof,which is sufficient to inhibit growth of the microorganisms in question.

Finally the invention also relates to wound healing composition orproducts such as bandages, medical devices such as, e.g., catheters andfurther to anti-dandruff hair products, such as shampoos.

Materials and Methods

Materials:

-   A. niger strain FGSC A798 from the Fungal Genetic Stock Center, U.    of Kansas Medical Center.-   A. niger MBin118 is A. niger, BO-1 (DSM 12665) (which is disclosed    in WO 00/50576) derivative in which the alpha-amylase genes, the    glucoamylase gene, the acid stable amylase gene and the prtT gene    have been deleted.-   Bech 2 is a cyclopiazonic acid (CPA) negative and kojic acid (KA)    negative Aspergillus oryzae strain. The construction of Bech 2 is    described in Example 1 of WO 00/39322.-   pMT2188 is based on Aspergillus expression plasmid pCaHj527    (WO 0070064) constructed as described in Example 7 of Danish patent    application PA 2001 00088 (published as WO 02/12472).    Methods:    Cultivation of an A. niger Strain Producing the Anti-Microbial    Polypeptide

The production and subsequent purification of the anti-microbialpolypeptide shown in SEQ ID NO: 1 may be carried out as described in WO94/01459. In brief, the anti-microbial polypeptide is prepared asfollows:

Cultivation on agar slants is carried out using an agar medium preparedfrom 39 g of Potato Dextrose Agar (from Difco) and distilled water up to1000 ml. The agar slants are inoculated with the A. niger strain andgrown for one week at 26° C.

Submerged Cultivation

A 500 ml Erlenmeyer flask containing 100 ml of AMC substrate (15 g meatextract, 20 g Peptone, 20 g corn starch, 5 g NaCl, 1 ml Pluronic, 1liter H₂O) is inoculated with 5 ml of a spore suspension containing 10⁶spores/ml prepared from an agar slant culture of A. niger obtained asdescribed above containing 10⁶ spores per ml. The flask is shaken at 220rpm at 30° C. for 3 days, after which the polypeptide can be recovered.

Isolation, Purification and Amino Acid Sequence Determination of theAnti-Microbial Polypeptide

The fermentation broth obtained as described above is subjected tocentrifugation, the mycelium is suspended in Tris-buffered saline at pH7 and then subjected to a second centrifugation. The supernatants fromthe two centrifugations are combined and subjected to sterilefiltration. The pH of the resulting supernatant preparation is adjustedto between 6.5 and 9 and the supernatant preparation is applied to acation exchange resin (S Sepharose Fast Flow), which prior toapplication is equilibrated with a phosphate buffer at pH 6.5. Elutionof active fractions from this resin is accomplished by the applicationof a buffer with a high ionic strength such as a buffer comprising 20 mMphosphate, 1.5 mM NaCl, pH 6.5. If necessary, the procedure is repeatedafter dilution with or dialysis against a buffer with ionic strengthbelow or near that of 10 mM phosphate at pH 6.0. Elution from thissecond step is, if necessary, carried out as a gradient elution. Thepurity of the active fractions is assessed by HPLC.

The combined active fractions are subjected to sterile filtration on a0.22 micro m filter (Millipore) prior to testing of the anti-microbialproperties.

The purified Anti-microbial polypeptide is S-carboxymethylated using themethod described by Nakaya et al. (1990), Eur. J. Biochem. 19, 31-38,and de-salted using reverse phase HPLC. The S-carboxymethylatedanti-microbial polypeptide is subjected to N-terminal amino acidsequence determination on an Applied Biosystems 473A sequencer operatedin accordance with the manufacturer's instructions.

Extinction Coefficient

The extinction coefficient of the anti-microbial polypeptide isestimated from the amino acid sequence shown in SEQ ID NO: 2 using theformula

${ɛ^{0.1\%}\left( {280\mspace{14mu}{nm}} \right)} = \frac{{5690\; \cdot \left( {{No}.\mspace{14mu}{Trp}} \right)} + {1280\; \cdot \left( {{No}.\mspace{14mu}{Tyr}} \right)} + {120\; \cdot \left( {{No}.\mspace{14mu}{Cys}} \right)}}{{molecular}\mspace{14mu}{mass}}$(Gill & von Hippel, 1989), where (No. Trp), (No. Tyr) and (No. Cys) arethe number of Trp, Tyr and Cys residues in the amino acid sequence.

Based on this formula the extinction coefficient at 280 nm may becalculated. All polypeptide determinations are based on OD₂₈₀measurements using this calculated extinction coefficient.

In vitro Analysis of Anti-Microbial Activity

The sensitivity of filamentous fungi and bacteria towards anti-microbialpolypeptides may be tested using the below Bioassay I.

The petridishes used for assaying each of the bacteria and fungi aremade from agar prepared as follows:

Aspergillus oryzae (26□C), Aspergillus niger (26° C.):

2-6 ml of a suspension prepared from an agar slant culture containing10⁶ spores per ml are mixed with 100 ml of YPG-1-agar (yeast extract(0.4%), KH₂PO₄ (0.1%, MgSO₄, 7 H₂O, glucose (1.5%), Agar (1.5%), (48°C.).

Bacillus subtilis (30□C), Saccharomyces cerevicieae (26° C.):

A suspension prepared from an agar slant culture was suitably diluted sothat a mixture of 6 ml of the diluted suspension and 100 ml of anAntibiotic medium-1 (Difco) (48° C.) contained 10⁶ spores per ml.

Bioassay I

The assay is carried out in petridishes (14 cm), each prepared from 35ml of an agar suspension prepared as described above. In 4 mm holes madein the agar, 15 micro l of purified protein solution, the fermentedbroth and the sterile filtrate obtained as described above are applied.Plates containing bacteria are incubated for one day at the temperaturesindicated in the list of bacteria. Plates containing fungi are incubatedfor two days at 26° C.

The inhibition zone (in mm) in the petridishes is used to quantitativelymeasure the activity of the polypeptide in question against selectedfungi, yeasts and bacteria.

EXAMPLES Example 1

Cloning of an A. niger Anti-Microbial Polypeptide Encoding Gene

Six degenerate primers were designed from the peptide sequence given byLee et al (1999) Biochemical and Biophysical Research Communications,Vol. 263, No. 3, p. 646-651. The degeneration was used to cover mostpossibilities at the 3′end of the primers while the 5′ is ends werechosen on the basis of Aspergillus codon use.

The three forward primers were made:

92: CCAAGTACGGYGGYGARTG, (SEQ ID NO: 3) 93: CACAACACYTGYACYAAYTA, (SEQID NO: 4) and 94: AAGGAYGGYAAGAAYCAYGT. (SEQ ID NO: 5)

The three reverse primers were made:

95: ACGGTCTTGTGRTGRTCRTC, (SEQ ID NO: 6) 96: GTCGTCGTACTCRCARTGRTG, (SEQID NO: 7) and 97: GAGTGGTGGCGRTCRGTYTTRTG. (SEQ ID NO: 8)

Chromosomal DNA was prepared from Aspergillus niger strain C-40-1. PCRwith the nine relevant primer combinations were run on the fourchromosomal DNAs. The PCR was run for 35 cycles and the experiment wasrun both with annealing temperature of 45° C. and of 50° C., i.e., atotal of 72 PCR reactions.

The PCR products were separated on a 4% agarose gel. Due to thedegenerate primers used, quite a few small PCR products had beengenerated in virtually all of the PCR reactions. Four potentiallyinteresting band from A. niger C-40-1 PCRs were isolated and cloned intopCR4-TOPO T/A (purchased from Invitrogen, US). Particularly anapproximately 190 bp fragment from the primer combination 93/95 and a180 bp fragment from the combination 93/96 seemed promising. Sequencingof these pCR4 clones confirmed the two inserts to span a putative introntwo. PCR amplification on genomic DNA from Aspergillus niger FGSC A798using the 93/95 primer combination yielded a PCR fragment of identicallength and sequence as that obtained with the strain C-40-1 genomic DNA.

The PCR93/95 insert was used as a probe in Southerns of A. niger C-40-1genomic DNA. Not all digests appeared to be complete but the probe wasdefinitely found to hybridize to a 1.8 kb Pst1 fragment, a 2.7 kb Hind3fragment and a 4.8 kb EcoR1 fragment all of which were cloned by inversePCR using primers from the originally cloned 190 bp fragment. These PCRswere done with the proofreading Expand PCR kit and the products werecloned into pCR4TOPOZeroBlunt (purchased from Invitrogen, US).

Example 2

Expression of the A. niger Anti-Microbial Polypeptide

The A. niger anti-microbial polypeptide was inserted in the Aspergillusexpression vector pMT2188 to give pMT2446 (i.e., pMT2188 with an insertcomprising the A. niger anti-microbial gene): pMT2446 was transformedinto A. oryzae BECh2 (disclosed, e.g., on page 26 of WO 00/39322) andinto A. niger MBin118. Thirty transformants of each strain werere-isolated twice under selective and non-inducing conditions on Coveminimal plates with sucrose and acetamide. To test expression of theanti-microbial polypeptide, transformants were grown for 6 days in tubeswith 10 ml YPM (2% maltose). Supernatants were run on NuPage 10%Bis-Tris SDS gels with MES running buffer to allow separation in the lowm_(w) range.

It was noted that growth on YPM of several of the A. oryzae BECh2transformants seemed inhibited while the A. niger MBin118 transformantsseemed to grow normally.

Growth was inhibited in both A. oryzae BECh2 and A. niger MBin118transformed with an expression plasmid for the A. giganteus anti-fungalpolypeptide (AFP) disclosed in WO 94/01459).

Example 3

Purification and Characterisation of Anti-Microbial Peptide (AMP) fromAspergillus niger Strain MT2464

The broth from a fermentation of strain MT2464 (A. niger MBin118carrying the pMT2446 plasmid) was centrifuged at 10000 rpm, 4° C. for 15minutes. The supernatant was passed through a 0.22 micro m DuraporeMembrane filter (Millipore). 1.0 L of 0.22 micro m filtered supernatantwas added 5.0 L Milli-Q filtered H₂O and adjusted to pH 6.0. Theconductivity of the diluted supernatant was measured to 4.4 mSi/cm.

The diluted supernatant was loaded onto a 175 ml SP-Sepharose (PharmaciaBiotec) cat ion exchange column on a Pharmacia FPLC system and washedwith a buffer A (10 mM NaPO₄, pH 6.0). Elution was performed by runninga linear salt gradient obtained by mixing buffer A and B (10 mM NaPO₄,1.0M NaCl pH 6.0) increasing the fraction of B from 0 to 100% over 7column volumes. The flow rate was 10 ml/min. The MT2464-AMP elutedaround 35% B. This purification resulted in a single band on SDS PAGErunning around 7 kDa in agreement with the expected mass of theMT2464-AMP at 6511 Da.

N-Terminal Sequencing and Mass Spectrometry

The identity of the polypeptide product was confirmed by N-terminalsequencing.

10 microL sample was loaded directly onto a Micro TFA filter/PerkinElmer that was placed in the cartridge of an Applied Biosystems Prociseprotein sequencer. The N-terminal sequencing was carried out using themethod run file for Pulsed-Liquid. The following N-terminal sequence wasobtained: LSKYGGECSVEHNT (SEQ ID NO: 9)

The sequence is identical to the expected N-terminal of MT2464-AMP.

Furthermore the mass of the purified MT2464-AMP was determined on aVoyager MALDI-TOF (Applied Biosystems) usingalpha-Cyano-4-hydroxycinnamic acid as matrix. The mass was found to be6511 Da as expected from the sequence.

Example 4

Fermentation of A. niger Transformant

Cultivation of Strain MT2467 (A. niger MBin118 Carrying the pMT2446Plasmid)

Slant containing spores was used to inoculate a shake flask containing200 mL MLC medium. The shake flask was placed in an incubator at 30°C./250 rpm for approximately 24 hours. After this point the content ofthe shake flask was pumped into a 2 L (working volume) fermentor. 44hours after inoculation of the fermentor, addition of Feed medium wasstarted, and during the rest of the cultivation, the feed rate was keptconstant. The cultivation was run for a total of 165.8 hours, duringwhich the last approx. 100 hours were characterized by a low oxygentension and excess glucose in the fermentation broth.

Cultivation conditions for cultivation AnAFP-02 Pre-culture mediumGlucose * H₂O   40 g/L Soy meal   50 g/L Citric acid   4 g/L PluronicPE6100  0.1 mL/L Main Tank medium Glucose * H₂O  100 g/L (NH₄)₂HPO₄(separate autoclavation)  5.0 g/L MgSO₄, 7H₂O  2.0 g/L KH₂PO₄  2.0 g/LTrace metals 0.25 mL/L Pluronic   1 mL/L Feed medium Glucose * H₂O  351g/L Pluronic   2 mL/L Other cultivation conditions Volume of medium inthe batch phase  2.0 L Temperature   34° C. pH  5.3 Aeration   2 L/minFeed start criteria   44 hours after inoculation Feed rate   10 g/hourpH was regulated by adding 10% (w/w) NH₃ or 10% (w/w) H₃PO₄; thecultivation tanks were inoculated with the content of a 200 mL shakeflask that had been incubated at 30□C for approximately 24 hours.Determination of A. niger Anti-Microbial Polypeptide Yield Using theLC-MS Method

Precise determination of the concentration of the purified MT2464-AMPwas achieved by Amino acid analysis. A sample was hydrolysed byincubation in 6M HCl and 0.1% phenol under vacuum at 110° C. for 16hours. The hydrolysate was analysed on a 420A amino acid analyzer(Applied Biosystems). The concentration of MT2464-AMP was determined to8.15 mg/ml. This concentration was used as standard and used as basisfor the LC-MS based quantification method.

Running the mass spectrometer, a Hewlett Packard 1100 series MSD, in thescan mode, a diluted sample from this standard gave one major peak. Themass spectrum of this peak showed five prominent ions, 931.2 Da, 1085.9Da, 1303.2 Da, 1628.8 Da, and 2171.4 Da, which by deconvolutioncorrespond to a peptide with a mass of 6510 Da. The quantification wascarried out by selected ion monitoring (SIM mode) of the ions at 1086Da, 1303 Da and 1629 Da. The column was a C4 Aquapore BU-300, 7 micro m,30×3.2 mm from Perkin Elmer, and the eluents were A: 3% acetonitril and0.1% trifluoroacetic acid; and B: 95% acetonitril and 0.085%trifluoroacetic acid. The flow rate was 0.25 mL pr. minute and a lineareluent gradient going from 0% to 50% B-eluent in 30 minutes was applied.

A standard curve was made (see FIG. 2) and used to determine theconcentration of the A. niger AMP (see FIG. 3). Based on this the A.niger anti-microbial polypeptide (AMP) yield was determined to be above2.0 g/L.

1. An isolated polypeptide exhibiting anti-microbial activity, which (a)is encoded by a DNA sequence which is at least 95% identical to the partof SEQ ID NO:1 encoding the mature anti-microbial polypeptide, or (b)comprises the amino acid sequence from position 1 to position 58 shownin SEQ ID NO:2.
 2. The polypeptide of claim 1, wherein theanti-microbial peptide has an N-terminal extension of 1-50 amino acids.3. The polypeptide of claim 1, wherein the anti-microbial peptide has anN-terminal extension of 2-20 amino acids.
 4. The polypeptide of claim 1,wherein the anti-microbial peptide has an N-terminal extension of 3-15amino acids.
 5. The polypeptide of claim 2, wherein the N-terminalextension does not contain an Arg (R).
 6. The polypeptide of claim 2,wherein the N-terminal extension comprises a kex2 or kex2-like cleavagesite.
 7. The polypeptide of claim 2, wherein the N-terminal extensioncomprises at least two E and/or D amino acid residues.
 8. Thepolypeptide of claim 2, wherein the N-terminal extension comprises oneof the following sequences: EAE, EE, DE, DD.
 9. The polypeptideaccording to claim 1, which is obtained from a microorganism.
 10. Thepolypeptide of claim 9, wherein the polypeptide is obtained fromAspergillus.
 11. The polypeptide of claim 10, wherein the polypeptide isobtained from A. aculeatus, A. awamori, A. clavatonanicus, A. clavatus,A. foetidus, A. giganteus, A. japonicus, A. longivesica, A. niger, A.pallidus, A. parasiticus, A. phoenicis, A. rhizopodus, and A. saitoi.12. The polypeptide of claim 11, wherein the polypeptide is obtainedfrom A. niger.
 13. The polypeptide of claim 1, which comprises the aminoacid sequence from position 1 to position 58 shown in SEQ ID NO:
 2. 14.An anti-microbial composition comprising an anti-microbial polypeptideof claim 1 and an additional biocidal agent.
 15. A method of controllingor combating microorganisms, comprising applying an anti-microbialpolypeptide claim 1 to the microorganisms.
 16. The polypeptide of claim1, wherein the polypeptide is encoded by a DNA sequence which is atleast 97% identical to the part of SEQ ID NO:1 encoding the matureanti-microbial polypeptide.
 17. The polypeptide of claim 1, wherein thepolypeptide is encoded by a DNA sequence which is at least 99% identicalto the part of SEQ ID NO:1 encoding the mature anti-microbialpolypeptide.